!Project Abstract The highly conserved abundant molecular chaperone Hsp90 is a global cellular regulator that interacts with client proteins in a dynamic ATP-dependent cycle to ensure client protein folding, transport and/or assembly into multiprotein complexes. Hsp90- dependent proteins have critical roles in many forms of cancer and neurodegenerative disease as well as cystic fibrosis and other diseases. Our long-term goal is to understand how Hsp90 and cochaperones cooperate in the folding of hundreds of proteins with diverse sequences and structures sufficiently to develop small compounds that only affect subsets of Hsp90 clients. In the first Aim, we will use a novel set of Hsp90 mutants to understand how three cochaperones that bind the closed, ATP and client-bound conformation of Hsp90 fine-tune client folding. The technically innovative second Aim will identify and characterize Hsp90 mutants that affect different subsets of client proteins. Together these studies will enable a better mechanistic understanding of the function of cochaperones in regulating client selection, conformation and activity. Identification of clusters of Hsp90 mutants with similar effects will pave a path towards rational design of compounds with selective effects on Hsp90 clients in vivo. !